Dong Maoqing, Hosohata Keiko, Pinon Delia I, Muthukumaraswamy Natesa, Miller Laurence J
Cancer Center and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona 85259, USA.
Mol Endocrinol. 2006 Jul;20(7):1688-98. doi: 10.1210/me.2006-0009. Epub 2006 Mar 2.
Understanding of the conformational changes in G protein-coupled receptors associated with activation and inactivation is of great interest. We previously used photoaffinity labeling to elucidate spatial approximations between photolabile residues situated throughout the pharmacophore of secretin agonist probes and this receptor. The aim of the current work was to develop analogous photolabile secretin antagonist probes and to explore their spatial approximations. The most potent secretin antagonist reported is a pseudopeptide ([psi(4, 5)]secretin) in which the peptide bond between residues 4 and 5 was replaced by a psi(CH(2)-NH) peptide bond isostere. We have developed a series of [psi(4, 5)]secretin analogs incorporating photolabile benzoyl phenylalanine residues in positions 6, 22, and 26. Each bound to the secretin receptor saturably and specifically, with affinity similar to their parental peptide. At concentrations with no measurable agonist activity, each probe covalently labeled the secretin receptor. Peptide mapping using proteolytic cleavage, immunoprecipitation, and radiochemical sequencing identified that each of these three probes labeled the amino terminus of the secretin receptor. Whereas the position 22 probe labeled the same residue as its analogous agonist probe and the position 6 probe labeled a residue within two residues of that labeled by its analogous agonist probe, the position 26 probe labeled a site 16 residues away from that labeled by its analogous agonist probe. Thus, whereas structurally related agonist and antagonist probes dock in the same general region of this receptor, conformational differences in active and inactive states result in substantial differences in spatial approximation at the carboxyl-terminal end of secretin analogs.
了解与激活和失活相关的G蛋白偶联受体的构象变化具有重大意义。我们之前使用光亲和标记来阐明分布在促胰液素激动剂探针整个药效基团中的光不稳定残基与该受体之间的空间近似性。当前工作的目的是开发类似的光不稳定促胰液素拮抗剂探针并探索它们的空间近似性。报道的最有效的促胰液素拮抗剂是一种假肽（[psi(4, 5)]促胰液素），其中第4和5位残基之间的肽键被psi(CH(2)-NH)肽键等排体取代。我们已经开发了一系列[psi(4, 5)]促胰液素类似物，在第6、22和26位引入了光不稳定的苯甲酰苯丙氨酸残基。每种类似物都以饱和且特异性的方式与促胰液素受体结合，亲和力与其亲本肽相似。在没有可测量的激动剂活性的浓度下，每种探针都共价标记了促胰液素受体。使用蛋白酶解、免疫沉淀和放射化学测序进行的肽图谱分析确定，这三种探针中的每一种都标记了促胰液素受体的氨基末端。虽然第22位探针标记的残基与其类似的激动剂探针相同，第6位探针标记的残基与其类似的激动剂探针标记的残基在两个残基范围内，但第26位探针标记的位点与其类似的激动剂探针标记的位点相距16个残基。因此，虽然结构相关的激动剂和拮抗剂探针在该受体的同一大致区域对接，但活性和非活性状态下的构象差异导致促胰液素类似物羧基末端的空间近似性存在显著差异。
